Elucidating Polyphosphate Anion Binding to Lanthanide Complexes Using EXAFS and Pulsed EPR Spectroscopy.

Reversible anion binding to lanthanide complexes in aqueous solution has emerged as an effective method for anion sensing. Through careful design of the organic ligand, luminescent lanthanide complexes capable of binding biologically relevant anions in a bidentate or monodentate manner can be realized. While single-crystal X-ray diffraction analyses and NMR spectroscopy have revealed the structural geometry of several host-guest complexes, the challenge remains in designing preorganized lanthanide receptors with enhanced anion selectivity for broader applications in diagnostics and bioimaging. To address this challenge, innovative and complementary methods to investigate host-anion binding geometry are becoming increasingly important. Herein, we demonstrate the combined use of Eu L3-edge extended X-ray absorption fine structure (EXAFS) and electron paramagnetic resonance (EPR) spectroscopy to elucidate the binding of nucleoside phosphates (ATP, ADP, and AMP) to a cationic lanthanide complex. We establish that ATP unequivocally binds the lanthanide center in a bidentate manner in water, while ADP adopts both bidentate and monodentate modes, and AMP binds in a monodentate manner. This interdisciplinary approach provides deeper insight into lanthanide host-guest chemistry in solution, laying the groundwork for designing emissive probes that undergo specific anion-induced structural changes and elicit desired optical responses upon binding.

A lanthanide tag for a complementary set of pseudocontact shifts.

Pseudocontact shifts (PCS) generated by paramagnetic lanthanide ions deliver powerful restraints for protein structure analysis by NMR spectroscopy. We present a new lanthanide tag that generates different PCSs than that of a related tag, which differs in structure by a single oxygen atom. It is highly reactive towards cysteine and performs well in turn-on luminescence and in EPR spectroscopy.

A dual encapsulation strategy to generate anion-responsive luminescent lanthanide hydrogels.

We report a new method to generate ion-responsive luminescent hydrogels, involving encapsulation of a luminescent lanthanide probe within crosslinked amphiphilic polymer particles and subsequent entrapment within a hydrogel. The resulting hydrogels are capable of reversible bicarbonate sensing, exhibit no leaching, and can be tuned for a range of sensing applications.

Transmembrane Transport of Inorganic Phosphate by a Strapped Calix[4]pyrrole.

Synthetic anion receptors are increasingly being explored for the transport of anions across lipid membranes because of their potential therapeutic applications. A considerable amount of research focuses on the transport of chloride, whereas the transmembrane transport of inorganic phosphate has not been reported to date, despite the biological relevance of this anion. Here we present a calix[4]pyrrole with a bisurea strap that functions as a receptor and transporter for H2PO4-, relying on the formation of eight hydrogen bonds and efficient encapsulation of the anion. Using a phosphate-sensitive lanthanide probe and 31P NMR spectroscopy, we demonstrate that this receptor can transport phosphate into vesicles by H2PO4-/Cl- antiport, H2PO4- uniport, and Cs+/H2PO4- symport mechanisms. This first example of inorganic phosphate transport by a neutral receptor opens perspectives for the future development of transporters for various biological phosphates.

Structural and functional properties of the transporter SLC26A6 reveal mechanism of coupled anion exchange.

Members of the SLC26 family constitute a conserved class of anion transport proteins, which encompasses uncoupled transporters with channel-like properties, coupled exchangers and motor proteins. Among the 10 functional paralogs in humans, several participate in the secretion of bicarbonate in exchange with chloride and thus play an important role in maintaining pH homeostasis. Previously, we have elucidated the structure of murine SLC26A9 and defined its function as an uncoupled chloride transporter (Walter et al., 2019). Here we have determined the structure of the closely related human transporter SLC26A6 and characterized it as a coupled exchanger of chloride with bicarbonate and presumably also oxalate. The structure defines an inward-facing conformation of the protein that generally resembles known structures of SLC26A9. The altered anion selectivity between both paralogs is a consequence of a remodeled ion binding site located in the center of a mobile unit of the membrane-inserted domain, which also accounts for differences in the coupling mechanism.

Expedient synthesis and luminescence sensing of the inositol pyrophosphate cellular messenger 5-PP-InsP5.

Inositol pyrophosphates are important biomolecules associated with apoptosis, cell growth and kinase regulation, yet their exact biological roles are still emerging and probes do not exist for their selective detection. We report the first molecular probe for the selective and sensitive detection of the most abundant cellular inositol pyrophosphate 5-PP-InsP5, as well as an efficient new synthesis. The probe is based on a macrocyclic Eu(iii) complex bearing two quinoline arms providing a free coordination site at the Eu(iii) metal centre. Bidentate binding of the pyrophosphate group of 5-PP-InsP5 to the Eu(iii) ion is proposed, supported by DFT calculations, giving rise to a selective enhancement in Eu(iii) emission intensity and lifetime. We demonstrate the use of time-resolved luminescence as a bioassay tool for monitoring enzymatic processes in which 5-PP-InsP5 is consumed. Our probe offers a potential screening methodology to identify drug-like compounds that modulate the activity of enzymes of inositol pyrophosphate metabolism.

Transmembrane transport of fluoride studied by time-resolved emission spectroscopy.

Here we present a new method to monitor fluoride transmembrane transport into liposomes using a europium(III) complex. We take advantage of the long emission lifetime of this probe to measure the transport activity of a fluorescent transporter. The high sensitivity, selectivity, and versatility of the assay allowed us to study different types of fluoride transporters and unravel their mechanisms of action.

Dissecting transmembrane bicarbonate transport by 1,8-di(thio)amidocarbazoles.

Synthetic ionophores able to transport bicarbonate and chloride anions across lipid bilayers are appealing for their wide range of potential biological applications. We have studied the bicarbonate and chloride transport by carbazoles with two amido/thioamido groups using a bicarbonate-sensitive europium(III) probe in liposomes and found a highly remarkable transporter concentration dependence. This can be explained by a combination of two distinct transport mechanisms: HCO3-/Cl- exchange and a combination of unassisted CO2 diffusion and HCl transport, of which the respective contributions were quantified. The compounds studied were found to be highly potent HCl transporters. Based on the mechanistic insights on anion transport, we have tested the antimicrobial activity of these compounds and found a good correlation with their ion transport properties and a high activity against Gram-positive bacteria.

Monofunctionalized Fluorinated Bambusurils and Their Conjugates for Anion Transport and Extraction.

Bambusurils are macrocyclic molecules that are known for their high binding affinity and selectivity toward anions. Here, we present the preparation of two bambusurils bearing fluorinated substituents and one carboxylic function. These monofunctionalized bambusurils were conjugated with crown ether and cholesterol units. The resulting conjugates were successfully tested in liquid-liquid extraction of inorganic salts and chloride/bicarbonate transport across lipid bilayers.

Sterically demanding macrocyclic Eu(iii) complexes for selective recognition of phosphate and real-time monitoring of enzymatically generated adenosine monophosphate.

The design of molecular receptors that bind and sense anions in biologically relevant aqueous solutions is a key challenge in supramolecular chemistry. The recognition of inorganic phosphate is particularly challenging because of its high hydration energy and pH dependent speciation. Adenosine monophosphate (AMP) represents a valuable but elusive target for supramolecular detection because of its structural similarity to the more negatively charged anions, ATP and ADP. We report two new macrocyclic Eu(iii) receptors capable of selectively sensing inorganic phosphate and AMP in water. The receptors contain a sterically demanding 8-(benzyloxy)quinoline pendant arm that coordinates to the metal centre, creating a binding pocket suitable for phosphate and AMP, whilst excluding potentially interfering chelating anions, in particular ATP, bicarbonate and lactate. The sensing selectivity of our Eu(iii) receptors follows the order AMP > ADP > ATP, which represents a reversal of the order of selectivity observed for most reported nucleoside phosphate receptors. We have exploited the unique host-guest induced changes in emission intensity and lifetime for the detection of inorganic phosphate in human serum samples, and for monitoring the enzymatic production of AMP in real-time.

Anion binding to a cationic europium(III) probe enables the first real-time assay of heparan sulfotransferase activity.

Sulfotransferases constitute a ubiquitous class of enzymes which are poorly understood due to the lack of a convenient tool for screening their activity. These enzymes use the anion PAPS (adenosine-3'-phosphate-5'-phosphosulfate) as a donor for a broad range of acceptor substrates, including carbohydrates, producing sulfated compounds and PAP (adenosine-3',5'-diphosphate) as a side product. We present a europium(III)-based probe that binds reversibly to both PAPS and PAP, producing a larger luminescence enhancement with the latter anion. We exploit this greater emission enhancement with PAP to demonstrate the first direct real-time assay of a heparan sulfate sulfotransferase using a multi-well plate format. The selective response of our probe towards PAP over structurally similar nucleoside phosphate anions, and over other anions, is investigated and discussed. This work opens the possibility of investigating more fully the roles played by this enzyme class in health and disease, including operationally simple inhibitor screening.

Localising individual atoms of tryptophan side chains in the metallo-ß-lactamase IMP-1 by pseudocontact shifts from paramagnetic lanthanoid tags at multiple sites.

The metallo-ß-lactamase IMP-1 features a flexible loop near the active site that assumes different conformations in single crystal structures, which may assist in substrate binding and enzymatic activity. To probe the position of this loop, we labelled the tryptophan residues of IMP-1 with 7-13C-indole and the protein with lanthanoid tags at three different sites. The magnetic susceptibility anisotropy (Δχ) tensors were determined by measuring pseudocontact shifts (PCSs) of backbone amide protons. The Δχ tensors were subsequently used to identify the atomic coordinates of the tryptophan side chains in the protein. The PCSs were sufficient to determine the location of Trp28, which is in the active site loop targeted by our experiments, with high accuracy. Its average atomic coordinates showed barely significant changes in response to the inhibitor captopril. It was found that localisation spaces could be defined with better accuracy by including only the PCSs of a single paramagnetic lanthanoid ion for each tag and tagging site. The effect was attributed to the shallow angle with which PCS isosurfaces tend to intersect if generated by tags and tagging sites that are identical except for the paramagnetic lanthanoid ion.

Localising nuclear spins by pseudocontact shifts from a single tagging site.

Ligating a protein at a specific site with a tag molecule containing a paramagnetic metal ion provides a versatile way of generating pseudocontact shifts (PCSs) in nuclear magnetic resonance (NMR) spectra. PCSs can be observed for nuclear spins far from the tagging site, and PCSs generated from multiple tagging sites have been shown to enable highly accurate structure determinations at specific sites of interest, even when using flexible tags, provided the fitted effective magnetic susceptibility anisotropy (Δχ) tensors accurately back-calculate the experimental PCSs measured in the immediate vicinity of the site of interest. The present work investigates the situation where only the local structure of a protein region or bound ligand is to be determined rather than the structure of the entire molecular system. In this case, the need for gathering structural information from tags deployed at multiple sites may be queried. Our study presents a computational simulation of the structural information available from samples produced with single tags attached at up to six different sites, up to six different tags attached to a single site, and in-between scenarios. The results indicate that the number of tags is more important than the number of tagging sites. This has important practical implications, as it is much easier to identify a single site that is suitable for tagging than multiple ones. In an initial experimental demonstration with the ubiquitin mutant S57C, PCSs generated with four different tags at a single site are shown to accurately pinpoint the location of amide protons in different segments of the protein.

A Chiral Lanthanide Tag for Stable and Rigid Attachment to Single Cysteine Residues in Proteins for NMR, EPR and Time-Resolved Luminescence Studies.

A lanthanide-binding tag site-specifically attached to a protein presents a tool to probe the protein by multiple spectroscopic techniques, including nuclear magnetic resonance, electron paramagnetic resonance and time-resolved luminescence spectroscopy. Here a new stable chiral LnIII tag, referred to as C12, is presented for spontaneous and quantitative reaction with a cysteine residue to generate a stable thioether bond. The synthetic protocol of the tag is relatively straightforward, and the tag is stable for storage and shipping. It displays greatly enhanced reactivity towards selenocysteine, opening a route towards selective tagging of selenocysteine in proteins containing cysteine residues. Loaded with TbIII or TmIII ions, the C12 tag readily generates pseudocontact shifts (PCS) in protein NMR spectra. It produces a relatively rigid tether between lanthanide and protein, which is beneficial for interpretation of the PCSs by single magnetic susceptibility anisotropy tensors, and it is suitable for measuring distance distributions in double electron-electron resonance experiments. Upon reaction with cysteine or other thiol compounds, the TbIII complex exhibits a 100-fold enhancement in luminescence quantum yield, affording a highly sensitive turn-on luminescence probe for time-resolved FRET assays and enzyme reaction monitoring.

Polymerizable Gd(iii) building blocks for the synthesis of high relaxivity macromolecular MRI contrast agents.

A new synthetic strategy for the preparation of macromolecular MRI contrast agents (CAs) is reported. Four gadolinium(iii) complexes bearing either one or two polymerizable methacrylamide groups were synthesized, serving as monomers or crosslinkers for the preparation of water-soluble, polymeric CAs using Reversible Addition-Fragmentation Chain Transfer (RAFT) polymerization. Using this approach, macromolecular CAs were synthesized with different architectures, including linear, hyperbranched polymers and gels. The relaxivities of the polymeric CAs were determined by NMR relaxometry, revealing an up to 5-fold increase in relaxivity (60 MHz, 310 K) for the linear polymers compared with the clinically used CA, Gd-DOTA. Moreover, hyperbranched polymers obtained from Gd(iii) crosslinkers, displayed even higher relaxivities up to 22.8 mM-1 s-1, approximately 8 times higher than that of Gd-DOTA (60 MHz, 310 K). A detailed NMRD study revealed that the enhanced relaxivities of the hyperbranched polymers were obtained by limiting the local motion of the crosslinked Gd(iii) chelate. The versatility of RAFT polymerization of Gd(iii) monomers and crosslinkers opens the doors to more advanced polymeric CAs capable of multimodal, bioresponsive or targeting properties.

Advances in anion binding and sensing using luminescent lanthanide complexes.

Luminescent lanthanide complexes have been actively studied as selective anion receptors for the past two decades. Ln(iii) complexes, particularly of europium(iii) and terbium(iii), offer unique photophysical properties that are very valuable for anion sensing in biological media, including long luminescence lifetimes (milliseconds) that enable time-gating methods to eliminate background autofluorescence from biomolecules, and line-like emission spectra that allow ratiometric measurements. By careful design of the organic ligand, stable Ln(iii) complexes can be devised for rapid and reversible anion binding, providing a luminescence response that is fast and sensitive, offering the high spatial resolution required for biological imaging applications. This review focuses on recent progress in the development of Ln(iii) receptors that exhibit sufficiently high anion selectivity to be utilised in biological or environmental sensing applications. We evaluate the mechanisms of anion binding and sensing, and the strategies employed to tune anion affinity and selectivity, through variations in the structure and geometry of the ligand. We highlight examples of luminescent Ln(iii) receptors that have been utilised to detect and quantify specific anions in biological media (e.g. human serum), monitor enzyme reactions in real-time, and visualise target anions with high sensitivity in living cells.

Anion Receptors for the Discrimination of ATP and ADP in Biological Media.

The design and synthesis of molecular receptors for the selective binding of nucleoside phosphate anions (e. g. ATP, ADP, GTP, GDP, UDP) in aqueous media at physiological pH is a valuable research endeavour, which could lead to new sensing tools for biomedical and drug discovery research. However, this target is very challenging due to similarities in anion size, structure and charge. This Minireview provides an account of the development of receptors capable of discriminating between ATP and ADP, and their utilisation in biological sensing applications. Particular focus is given to the application of receptors for the determination of ATP or ADP concentrations in biological media, tracking ATP levels (or the ATP/ADP ratio) in cells using fluorescence microscopy, or real-time monitoring of enzyme reactions involving ATP and ADP in vitro.

Critical Evaluation of the Interaction of Reactive Oxygen and Nitrogen Species with Blood to Inform the Clinical Translation of Nonthermal Plasma Therapy.

Non-thermal plasma (NTP), an ionized gas generated at ambient pressure and temperature, has been an emerging technology for medical applications. Through controlled delivery of reactive oxygen and nitrogen species (ROS/RNS), NTP can elicit hormetic cellular responses, thus stimulating broad therapeutic effects. To enable clinical translation of the promising preclinical research into NTP therapy, a deeper understanding of NTP interactions with clinical substrates is profoundly needed. Since NTP-generated ROS/RNS will inevitably interact with blood in several clinical contexts, understanding their stability in this system is crucial. In this study, two medically relevant NTP delivery modalities were used to assess the stability of NTP-generated ROS/RNS in three aqueous solutions with increasing organic complexities: phosphate-buffered saline (PBS), blood plasma (BP), and processed whole blood. NTP-generated RNS collectively (NO2 -, ONOO-), H2O2, and ONOO- exclusively were analyzed over time. We demonstrated that NTP-generated RNS and H2O2 were stable in PBS but scavenged by different components of the blood. While RNS remained stable in BP after initial scavenging effects, it was completely reduced in processed whole blood. On the other hand, H2O2 was completely scavenged in both liquids over time. Our previously developed luminescent probe europium(III) was used for precision measurement of ONOO- concentration. NTP-generated ONOO- was detected in all three liquids for up to at least 30 seconds, thus highlighting its therapeutic potential. Based on our results, we discussed the necessary considerations to choose the most optimal NTP modality for delivery of ROS/RNS to and via blood in the clinical context.

Tuning the anion binding properties of lanthanide receptors to discriminate nucleoside phosphates in a sensing array.

The development of synthetic receptors for the selective binding and discrimination of anions in water requires an understanding of how anions interact with these synthetic receptors. Molecules designed to differentiate nucleoside phosphate anions (e.g. ATP, ADP, GTP, GDP, UDP) under physiological conditions could underpin exciting new sensing tools for biomedical research and drug discovery, but it is very challenging due to the similarities in anion structure, size and charge. We present a series of lanthanide-based anion receptors and establish key structural elements that impact on nucleoside phosphate anion binding and sensing. Structural evidence of anion binding using X-ray crystallographic and NMR data, supported by DFT calculations indicate the binding modes between the lanthanide complexes and certain phosphoanions, revealing a bidentate (α-, γ-) binding mode to ATP. We further use four of the receptors to allow discrimination of eight nucleoside phosphate anions in the first array-based assay using lanthanide complexes, taking advantage of the multiple emission bands and long emission lifetimes associated with luminescent lanthanide complexes.

Time-resolved luminescence detection of peroxynitrite using a reactivity-based lanthanide probe.

Peroxynitrite (ONOO-) is a powerful and short-lived oxidant formed in vivo, which can react with most biomolecules directly. To fully understand the roles of ONOO- in cell biology, improved methods for the selective detection and real-time analysis of ONOO- are needed. We present a water-soluble, luminescent europium(iii) probe for the rapid and sensitive detection of peroxynitrite in human serum, living cells and biological matrices. We have utilised the long luminescence lifetime of the probe to measure ONOO- in a time-resolved manner, effectively avoiding the influence of autofluorescence in biological samples. To demonstrate the utility of the Eu(iii) probe, we monitored the production of ONOO- in different cell lines, following treatment with a cold atmospheric plasma device commonly used in the clinic for skin wound treatment.